The axonal transport and regulated accumulation of neurofilaments (NF) are essential for axons to achieve the large calibers required for normal impulse conduction. NF accumulate abnormally as a pathologic hallmark of several major neurodegenerative diseases, which is believed to contribute to the neuronal dysfunction and degeneration in these diseases. We have identified novel features of NF transport in axons that may regulate axonal NF number. One aspect of regulation involves a NF phosphorylation sequence in axons which culminates in one specific phosphorylation event that is closely associated with profound changes in NF behavior and axonal morphology and is modulated by signals from myelinating glial cells. We propose to identify this site of phosphorylation on NFH and NFM subunits and define its regulation by protein kinases and phosphatases in vivo (Aim 1). We will test definitively the hypotheses that (a) phosphorylation of this site promotes the prolonged dissociation of NF from the transport mechanism and, thereby regulates the local accumulation of NF along axons, which, in turn, influences axon caliber; and (b) accentuation of this process leads to pathological NF accumulation relevant to human neurodegenerative disease pathogenesis (Aim 2). These multidisciplinary studies will emphasize in vivo approaches applied to existing and new mouse models of altered NF behavior created by gene targeting and gene replacement techniques. A second suspected determinant of NF accumulation is the rate of NF transport, which is governed by motile mechanisms that are still poorly understood. Recently, we have unequivocally identified the molecular motor Myosin V as a previously unrecognized major ligand of the core subunit of NF (NFL), the subunit required for NF transport. To clarify the role of Myosin V as a possible slow transport motor in vivo, we propose to identify the interacting polypeptide domains on NFL and Myosin V and to characterize the regulation of the interaction by phosphorylation. We will then characterize slow transport of NF and other proteins in mice in which normal NFL has been replaced with NFL lacking the Myosin V binding domain and in dilute-lethal mutant mice which carry a Myosin V mutation and produce no Myosin V protein (Aim 3). These studies address fundamental aspects of NF biology related to the normal functions of axons and are relevant to mechanisms of neuro-axonal degeneration in neurofibrillary diseases, including Alzheimer's disease and related dementias, amyotrophic lateral sclerosis, and glaucoma, as well as in demyelinating diseases, such as multiple sclerosis.